Determination of lower fatty acids, particularly the anti-epileptic dipropyl-acetic acid, in biological materials by means of micro diffusion and gas chromatography

A gas-liquid Chromatographic procedure with electron capture detection has been developed to measure the catecholamines, noradrenaline, adrenaline and dopamine in normal human urine as their pentafluoropropionyl derivatives. The amines are absorbed from urine at pH 8.5 on micro-columns of alumina, eluted with acetic acid and derivatized with pentafluoropropionic anhydride. Some improvements in the conventional procedure of chromatography on alumina have been introduced. Quantification is obtained by comparing peak heights with those of internal standards added to urine and taken through the entire determination.Normal urinary excretion values of free catecholamines (mean ± S.D.), expressed as μg per 24 h, were: noradrenaline, 52.1 ± 15.0, adrenaline, 24.2 ± 4.0 and dopamine 402.4 ± 86.3. Conjugated dopamine can be determined after acid hydrolysis (pH 1, 100°, 20 min). About 50 to 75% of total dopamine output is excreted in conjugated form.     

Influence of the parasympathetic and sympathetic nervous system on nocturnal bronchial obstruction

To determine whether an autonomic nervous system imbalance might underlie the nocturnal dyspnoea in patients with chronic airflow obstruction (CAO), we determined FEV1, sinus arrhythmia gap (SA gap), heart rate and urinary adrenaline and noradrenaline excretion every 4 h over 24 h. Measurements were performed in eight non-allergic patients with CAO and eight age- and sex-matched normal controls. The amplitude of the circadian changes in FEV1 in patients and controls was 27 +/- 2% and 7 +/- 1% respectively (P less than 0.001). Both an increased SA gap and a decreased heart rate are features of increased vagal activity. This vagal activity was significantly increased in patients, compared with normal controls (difference P less than 0.01), the difference being maximal at night. This increased activity might contribute to a bronchial obstruction in these patients. Urinary adrenaline excretion was significantly higher by day than by night in both patients and normal controls (P less than 0.01). The urinary levels of adrenaline in the patients were significantly decreased at all hours of observation as compared with levels in normal controls (P less than 0.05). Urinary noradrenaline levels were significantly lower in patients as compared with normal subjects (P less than 0.01), and lower by night than by day. Urinary histamine and Nt-methylhistamine excretion were in the normal range in each individual. Urinary levels, however, were significantly higher in patients at all hours of observation (P less than 0.05). No circadian rhythm was shown. Plasma cortisol levels showed a normal circadian variation, similar in patients and normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical, autonomic and therapeutic observations in two siblings with postural hypotension and sympathetic failure due to an inability to synthesize noradrenaline from dopamine because of a deficiency of dopamine beta hydroxylase

A brother and sister with long-standing symptoms of postural hypotension are described. They were considerably worse in the morning, after exercise and in warm weather. In the male, erection was unaffected but ejaculation was prolonged or absent. Both had nocturia, but there were no urinary bladder, bowel or sweating abnormalities. Autonomic function tests confirmed sympathetic adrenergic failure with spared sympathetic cholinergic and intact parasympathetic function. There were no other neurological abnormalities. Noradrenaline and adrenaline were undetectable in the plasma, but plasma dopamine was elevated. Urinary levels of noradrenaline and adrenaline metabolites were below detection limits, but dopamine metabolites were normal or elevated. Dopamine beta-hydroxylase activity was undetectable in the plasma. Immunohistochemical studies of perivascular cutaneous tissue confirmed normal peptidergic and tyrosine hydroxylase immunoreactivity, with absent dopamine beta-hydroxylase immunoreactivity. The findings were consistent with an enzymatic deficit in the conversion of dopamine to noradrenaline. The parents were clinically and biochemically normal. Treatment of both patients with the synthetic amino acid, d-l-threo-dihydroxyphenylserine, which contains a hydroxyl group and is converted to noradrenaline by dopa-decarboxylase, reduced symptoms and signs of postural hypotension and increased levels of plasma noradrenaline and its urinary metabolites. In the male, ejaculation became possible. Behavioural changes included a feeling of confidence and optimism, with a tendency to be argumentative. The laevo isomer also raised blood pressure and plasma noradrenaline levels. The drug had no direct pressor effects, as its actions were prevented by the dopa-decarboxylase inhibitor, carbidopa.     

Noradrenaline and Adrenaline in Blood and Urine in a Case of Phaeochromocytoma

A case of phaeochromocytoma is described in which X-ray investigation and pharmacodynamical tests with tetraethyl-ammonium and Regitin were non-informative. The diagnosis was established by the increased excretion of catechols with the urine (1010–2400 μg noradrenaline and 16–19 μg adrenaline per 24-hour period).

Estimation of catechols in samples of the blood and of the urine from the same period showed an average blood level of 3.6 μg/100 ml noradrenaline and a corresponding urinary output of 110 μg per hour.

The high systolic and diastolic blood pressure, the electrocardiographic changes, the increased basal metabolic rate, the response to the glucose tolerance test, sweating, eosinopenia and fatigue are explained as symptoms and signs of hypernoradrenalinemia.

Biological estimation of the catechols in the tumor, which weighed 40 g, showed 590 μg/g noradrenaline and 12 μg/g adrenaline.

After operation the blood pressure and the level of the urinary catechols returned to normal.     

Further studies on the mechanism of the natriuretic response to low-dose dopamine in man: effect on lithium clearance and nephrogenic cAMP formation

The effect of intravenous infusion of low-dose dopamine on electrolyte excretion, lithium clearance, nephrogenous cAMP formation and renal haemodynamics was investigated in healthy volunteers. Dopamine significantly increased the urine flow rate by 70.6% and urinary sodium excretion by 72%, but did not change creatinine clearance, PRA or plasma levels of AVP, ANP and cAMP. Renal plasma flow significantly increased by 48.6%; the glomerular filtration rate was not changed. Lithium per se increased basal PRA, but had no effect on the increments of urine flow rate, sodium excretion and renal blood flow induced by dopamine. Dopamine significantly increased the fractional excretion of lithium (representing fractional excretion of sodium at the proximal level). The increase in urinary sodium excretion during dopamine infusion, significantly correlated with the increase in fractional excretion of lithium (r = 0.94; P less than 0.01) and the increase in nephrogenous cAMP formation (r = 0.96; P less than 0.01). No correlation was found between the increase in urinary sodium excretion and the increase in renal blood flow. In conclusion, this study confirms that low-dose dopamine increases renal blood flow and urinary sodium excretion in healthy volunteers. This natriuretic response appears to be due to interaction with proximal tubular dopamine receptors, which are positively coupled to adenylate cyclase.     

The effect of urinary pH and flow rate on monoamine output.

Three-hour urinary output values of dopamine, noradrenaline, adrenaline, 2-phenylethylamine and p-tyramine were measured in normal volunteers who had been induced, by pretreatment with ammonium chloride or sodium bicarbonate, to excrete maximally acid or alkaline urine. There were significant effects on the excretion of dopamine, adrenaline and 2-phenylethylamine, inversely proportional to urinary pH value. Adrenaline output increased with increasing urinary flow rate. There was a significant correlation between urinary concentrations of 2-phenylethylamine and p-tyramine. These findings may have important clinical implications.     

Plasma and urine dopamine in man given sodium chloride in the diet.

1. Plasma and urine free dopamine were measured daily for 5 days in six normal subjects maintained on a low sodium diet. The subjects were then given dietary supplements of sodium chloride for 5 days and the measurements repeated. 2. Throughout the experiment the 24 h free dopamine excretion rates for all the subjects were higher than could be accounted for by renal clearance. Dopamine excretion increased significantly in response to the added sodium chloride whereas plasma dopamine remained unchanged. The rise in dopamine excretion preceded that of sodium excretion. 3. It is concluded that free dopamine is formed within the kidney in response to increased dietary sodium and may have a role in the control of sodium excretion.     

Clinical, Autonomic and Therapeutic Observations in Two Siblings With Postural Hypotension and Sympathetic Failure due to an Inability to Synthesize Noradrenaline From Dopamine Because of a Deficiency of Dopamine Beta Hydroxylase

A brother and sister with long-standing symptoms of posturalhypotension are described. They were considerably worse in themorning, after exercise and in warm weather. In the male, erectionwas unaffected but ejaculation was prolonged or absent. Bothhad nocturia, but there were no urinary bladder, bowel or sweatingabnormalities. Autonomic function tests confirmed sympatheticadrenergic failure with spared sympathetic cholinergic and intactparasympathetic function. There were no other neurological abnormalities.Noradrenaline and adrenaline were undetectable in the plasma,but plasma dopamine was elevated. Urinary levels of noradrenalineand adrenaline metabolites were below detection limits, butdopamine metabolites were normal or elevated. Dopamine ß-hydroxylaseactivity was undetectable in the plasma. Immunohistochemicalstudies of perivascular cutaneous tissue confirmed normal peptidergicand tyrosine hydroxylase immunoreactivity, with absent dopamineß-hydroxylase immunoreactivity. The findings wereconsistent with an enzymatic deficit in the conversion of dopamineto noradrenaline. The parents were clinically and biochemicallynormal. Treatment of both patients with the synthetic amino acid, d-l-threo-dihydroxyphenylserine,which contains a hydroxyl group and is converted to noradrenalineby dopa-decarboxylase, reduced symptoms and signs of posturalhypotension and increased levels of plasma noradrenaline andits urinary metabolites. In the male, ejaculation became possible.Behavioural changes included a feeling of confidence and optimism,with a tendency to be argumentative. The laevo isomer also raisedblood pressure and plasma noradrenaline levels. The drug hadno direct pressor effects, as its actions were prevented bythe dopa-decarboxylase inhibitor, carbidopa.     

Blood and urine histamine levels in normal and pathological states as measured by a radiochemical assay

The utility of the enzymatic radiochemical assay of histamine in diagnosing diseases with known abnormalities in histamine production was investigated. Whole blood histamine levels were abnormal only in patients with basophilia, i.e. chronic myelocytic leukemia or polycythemia vera. Histamine was not detectable (< 1 ng/ml) in normal plasma but was detected in plasma of some patients with either mastocytosis or chronic myelocytic leukemia. These patients also had symptoms which could be attributed to histamine release as, for example, hyperchlorhydria and hypotension. Urinary histamine excretion was also abnormally high in these diseases compared to normal subjects (range < 5–42 μg/24 h, $\text{n = 31}$). Patients with systemic mastocytosis had higher urine values (> 150 μg/24 h) than those with cutaneous mastocytosis (39–88 μg/ 24 h), and the urinary histamine excretion appeared to be an index of the severity of the disease. Studies with l-histidine loading suggest that the kidney is one possible source of urinary histamine.     

Effects of Dietary Sodium and of Acute Saline Infusion on the Interrelationship between Dopamine Excretion and Adrenergic Activity in Man

The effects of dietary sodium and of saline infusion on urinary dopamine and norepinephrine and on the relationship of these catecholamines to adrenergic activity were determined. In seven normal subjects on a 9-meq sodium intake, urinary dopamine and norepinephrine were 136+/-18 (SE) and 37.4+/-5.3 mug/day, respectively. When sodium intake was increased to 209 or 259 meq/day, urinary dopamine increased to 195+/-20 mug/day (P<0.01) whereas urinary norepinephrine decreased to 21.1+/-3.0 mug/day (P<0.01). Infusion of saline in seven subjects increased sodium excretion and urinary dopamine (from 2.18+/-0.22 to 2.79+/-0.19 mug/20 min, P<0.01), but decreased plasma dopamine-beta-hydroxylase by 33% and urinary norepinephrine insignificantly. The clearance of inulin and p-aminohippurate did not change significantly and filtration fraction was the same. The data indicate that an increase in dietary sodium or infusion of saline results in an apparent decrease in adrenergic activity and an increase in urinary dopamine. Dopamine excretion would thus appear to relate inversely to adrenergic activity and to parallel sodium excretion. These findings suggest a possible role for dopamine and norepinephrine in the regulation of renal sodium excretion.     

Inter-relationship between urinary kallikrein-kinins and arginine vasopressin in man

1. Physiological saline solution was infused in nine normal subjects and six patients with central diabetes insipidus (DI). At 120 min after the start of infusion, arginine vasopressin (AVP) was injected intramuscularly. Urine was collected in 30 min fractions before and after AVP administration. 2. The urinary excretions of kallikrein-like activity (KAL-A) (S-2266 hydrolysis activity) and immunoreactive kinins (i-kinins) were significantly lower in patients with DI than in normal subjects before AVP administration, while there were no differences in plasma renin activity, plasma aldosterone concentration, creatinine clearance and blood pressure between the two groups, except for a marked water diuresis in patients with DI. The urinary excretion of KAL-A and i-kinins correlated positively with the urinary excretion of AVP. 3. AVP administration increased both plasma AVP and urinary excretion of AVP to similar levels in both groups. As a result, urine volume decreased to a greater degree in patients with DI than in normal subjects. In contrast, the urinary excretions of KAL-A and i-kinins were increased by AVP administration, with a greater response in normal subjects than in the patients with DI. 4. After overnight fasting, acute water loading was carried out orally for 15 min in six normal subjects. At 30 min plasma AVP was suppressed by water loading to almost the basal level found in patients with DI. Urinary excretions of KAL-A and i-kinins in the first 30 min fraction after loading were also suppressed to the basal level in patients with DI. Later, the urinary excretion of KAL-A increased together with the increase in urine flow. Urine volume and free water clearance markedly increased except in the first 30 min fraction, compared with the control period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary excretion of aquaporin-2 in pathological states of water metabolism

Aquaporin-2 (AQP-2) is an arginine vasopressin (AVP)-regulated water channel in renal collecting duct cells. Approximately 3 % of AQP-2 in collecting duct cells is excreted into urine. Urinary excretion of AQP-2 varies widely in different physiological conditions, and it has a positive correlation with plasma AVP levels. Urinary excretion of AQP-2 was significantly increased by the single injection of AVP in patients with central diabetes insipidus. The urinary excretion of AQP-2 was one-eighth over in patients with central diabetes insipidus and three times greater in patients with impaired water excretion than that in normal subjects. In a hypertonic saline test, the urinary excretion of AQP-2 promptly increased 6-12-fold in normal subjects, but remained low in patients with central diabetes insipidus. In addition, exaggerated urinary excretion of AQP-2 persisted after an acute water load in patients with impaired water excretion. These results indicate that urinary excretion of AQP-2 is a potent marker for the diagnosis of water metabolism disorders dependent on AVP.     

Quantitative determination of serum and plasma haemoglobin using phenothiazines

A reliable high-performance liquid Chromatographic method is presented for the determination of the urinary free catecholamines noradrenaline, adrenaline and dopamine. Urine is purified on a column of immobilized boric acid. Catecholamines are separated by ion-pair reversed phase high-performance liquid chromatography and detected electrochemically.The method is suited for routine analysis. It allows the determination of urinary free catecholamines in concentrations as low as 1 μg/l for noradrenaline and adrenaline and 5 μg/l for dopamine. A single analysis can be completed within 1 h. Routine analyses can be carried out in a series of 40 samples within 2 days. The within-assay and between-assay coefficients of variation of the analyses in urine were both 2.9% for noradrenaline, both 5.0% for adrenaline, and 1.9 and 2.1% for dopamine.The Chromatographic properties of the immobilized boric acid were investigated. In particular, the elution pattern of a series of catecholamine metabolites and analogues was determined. Under the conditions used, only basic compounds containing both a vicinal hydroxyl configuration and a primary or secondary amino group adsorb and elute together with the free catecholamines.     

Plasma concentrations of adrenaline,noradrenaline and dopamine during forearm dynamic exercise

Summary. In six healthy volunteers plasma concentrations of adrenaline, noradrenaline and dopamine were measured at rest and during dynamic forearm exercise at submaximal and maximal intensities. Arterial and venous concentrations of adrenaline and noradrenaline increased with forearm exercise at all workloads. Dopamine concentrations did not change. The increases in adrenaline and noradrenaline were almost linearly related to the increase in heart rate with no levelling off at maximal exercise intensities. It is concluded that dynamic exercise with the forearm muscle group causes a small but significant activation of the sympatho-adrenal system as reflected by increases in plasma concentrations of adrenaline and noradrenaline.     

The urinary excretion of catecholamines and their derivatives in primary hypertension in man

1. The 24 h urinary excretion of adrenaline, noradrenaline, normetadrenaline, metadrenaline and vanilloylmandelic acid has been compared in 17 male normotensive subjects and 25 age-matched male hypertensive patients studied under similar in-patient conditions. 2. 24 h urinary metadrenaline was significantly lower in the hypertensive patients. With this exception, no significant differences were found between the two groups when the total 24 h excretion of free catecholamines and their metabolites was analysed. 3. Diurnal variation in free catecholamine excretion was found in both normotensive and hypertensive subjects. There was no corresponding variation in metabolite excretion. 4. No correlation could be established between systolic or diastolic blood pressure and the amounts of the catecholamines or their metabolites in the urine of either group. 5. The results are considered in the light of recent work demonstrating high plasma catecholamine concentrations in hypertension. They lend no support to the concept that excessive circulating catecholamines are responsible for the elevated blood pressure in essential hypertension.     

Abnormal plasma noradrenaline response and exercise induced albuminuria in type 1 (insulin-dependent) diabetes mellitus

Submaximal exercise provokes an abnormal elevation in albuminuria in type 1 (insulin-dependent) diabetes mellitus. Plasma catecholamines might be involved in this phenomenon by a renal vasoconstrictive effect. Twelve healthy subjects (Controls: albuminuria < 10 μg min^-1), 13 normoalbuminuric type 1 diabetic patients (DNormo: albuminuria < 10μg min^-1) and 13 microalbuminuric type 1 diabetic patients (DMicro: albuminuria 10–200 μg min^-1) performed a fixed bicycle workload (600 kpm for 20 min + urine collection 40 min post exercise). None of the patients suffered from autonomic neuropathy or hypertension. Fractional albumin clearance (FalbCl) rose in DNormo (p=0.02) and DMicro (p=0.01) but not in the Controls (p=0.40). Basal plasma adrenaline and noradrenaline were not different in the three groups. The increments in noradrenaline were more pronounced in DNormo and DMicro than in Control (Controls < DNormo, p < 0.05; Controls < DMicro, p < 0.01). The changes in FalbCl were significantly correlated with the changes in noradrenaline (all subjects r=0.65, p < 0.001). The increments in adrenaline were not different in the diabetic groups compared to the controls, and were not related to the changes in FalbCl. Multiple regression analysis showed that changes in plasma noradrenaline (p < 0.002) and in mean arterial pressure (p < 0.005) independently contributed to the changes in FalbCl (multiple r=0.73).

It is concluded that the exercise-induced plasma noradrenaline response is increased in normo-and microalbuminuric type-1 diabetic patients. Noradrenaline appears to contribute in the exercise-induced changes in renal protein handling, possibly by its effect on renal haemodynamics.     

The renin-aldosterone system in exaggerated natriuresis of essential hypertension

1. The effect of intravenous loading with 500 ml of sodium chloride solution (50 g/l) on plasma renin concentration, plasma aldosterone concentration, urinary sodium excretion and mean blood pressure was studied in 15 young patients with mild essential hypertension and 10 healthy normotensive control subjects. 2. Plasma renin concentration and plasma aldosterone concentration were suppressed to the same degree during loading in both the hypertensive and normotensive groups. Urinary sodium excretion was significantly higher in the hypertensive patients than in the normotensive subjects. Mean blood pressure increased slightly in both groups. 3. Plasma renin concentration and plasma aldosterone concentration were significantly correlated in both groups before sodium loading. The increase in urinary sodium excretion was significantly correlated to the suppression of plasma aldosterone concentration in the hypertensive, but not in the normotensive, group. No correlation was found between changes in urinary sodium excretion and changes in plasma renin concentration or mean blood pressure. 4. The results indicate that the suppressibility of the renin-aldosterone system by hyperosmotic sodium chloride solution is normal in young patients with mild essential hypertension. It is suggested that the changes in plasma aldosterone concentration induced by sodium loading might be involved in the regulation of exaggerated natriuresis in essential hypertension.     

Is dopamine a physiological natriuretic hormone in the dog?

Both plasma and urinary dopamine and noradrenaline were measured as free and sulphate conjugates, by a radioenzymatic method, before and during extracellular volume expansion (ECVE) with hypo-, iso- or hyper-tonic fluid (usually sodium chloride solution) in dogs. During ECVE there was a decrease in plasma catecholamine concentration. For all cases except noradrenaline, this is probably due to a dilution phenomenon since when results were expressed as pg/mg of protein, ECVE had no effect. This change in noradrenaline accounted for the increase in the dopamine/noradrenaline ratio. As expected, there was an increase in the urinary excretion of dopamine during ECVE with both iso- and hyper-tonic fluid. This increase was not observed in the group of dogs given hypotonic fluid, although the increase of fractional excretion of sodium was of a similar order of magnitude. The increase in the urinary excretion of dopamine was apparently not affected by an increase in plasma sodium concentration and/or osmolality. The demonstrated dissociation between sodium and dopamine in urine does not support a physiological role for dopamine in renal handling of sodium during ECVE, and raises the question of its specificity.